Team:Bielefeld-Germany/Protocols/Materials
From 2011.igem.org
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Materials: These enzymes, kits and materials were used in our project.
Used enzymes
Enzyme | Producer |
---|---|
AgeI | [http://www.fermentas.de/product_info.php?info=p247 Fermentas] |
DpnI | [http://www.fermentas.de/product_info.php?info=p296 Fermentas] |
EcoRI | [http://www.fermentas.de/product_info.php?info=p336 Fermentas] |
GoTaq DNA-polymerase | [http://www.promega.com/products/pcr/routine-pcr/gotaq-pcr-core-systems/ Promega] |
KOD Hotstart DNA-polymerase | [http://www.merck-chemicals.com/germany/life-science-research/kod-hot-start-dna-polymerase/EMD_BIO-71086/p_iFCb.s1O874AAAEj2Bl9.zLX Novagen] |
NgoMIV | [http://www.neb.com/nebecomm/products/productR0564.asp NEB] |
OneTaq DNA-polymerase | [http://www.neb.com/nebecomm/products/productM0480.asp NEB] |
Pfu DNA-polymerase | [http://www.promega.com/products/pcr/routine-pcr/pfu-dna-polymerase/ Promega] |
PstI | [http://www.fermentas.de/product_info.php?info=p458 Fermentas] |
Phusion HF DNA-polymerase | [http://www.finnzymes.com/pcr/phusion_high_fidelity_dna_polymerase.html Finnzymes] |
Shrimp alcaline phosphatase | [http://www.fermentas.de/product_info.php?info=p592 Fermentas] |
SpeI | [http://www.fermentas.de/product_info.php?info=p202 Fermentas] |
T4-DNA-Ligase | [http://www.fermentas.de/product_info.php?info=p580 Fermentas] |
T5 exonuclease | [http://www.neb.com/nebecomm/products/productM0363.asp NEB] |
taq DNA Ligase | [http://www.neb.com/nebecomm/products/productM0208.asp NEB] |
taq DNA-polymerase | [http://www.bioline.com/h_prod_detail.asp?itemid=219 Bioline] |
XbaI | [http://www.fermentas.de/product_info.php?info=p247 Fermentas] |
Used Kits
Function | Name |
---|---|
Molecular Cloning | [http://www.fermentas.de/product_info.php?info=p1620 Fermentas CloneJET™ PCR Cloning Kit] |
Plasmid purification | [http://www.fermentas.de/product_info.php?info=p874 Fermentas GeneJET™ Plasmid Miniprep Kit] |
Plasmid purification | [http://www.promega.com/b/de/minis/default.aspx?utm_source=Promega&utm_medium=Online&utm_campaign=Online_DEPureYield100Preps_Promega_HP_Banner Promega PureYield™ Plasmid Preps] |
PCR Cleanup | [http://www.mn-net.com/tabid/10745/default.aspx Macherey Nagel NucleoSpin® Extract II] |
PCR Cleanup | [http://www.promega.com/products/dna-and-rna-purification/dna-fragment-purification/wizard-sv-gel-and-pcr-clean_up-system/ Promega Wizard® SV Gel and PCR Clean-Up] |
PCR core system | [http://www.promega.com/products/pcr/routine-pcr/gotaq-pcr-core-systems/ Promega GoTaq® PCR Core System I] |
Media, buffer, solutions etc.
Ampicillin stock solution
- Solubilize 100 mg mL-1 Ampicillin
- Store at -20 °C
Chloramphenicol stock solution
- Solubilize 20 mg mL-1 Chloramphenicol in 100 % Ethanol
- Store at -20 °C
TAE buffer
For 1 L of 50 x TAE buffer you need:
- 242.48 g Tris
- 41.02 g Sodiumacetate
- 18.612 g EDTA
- Adjust pH to 7.8 with acetic acid
- Solve in dH2O
10 mL of the stock is diluted in 1 L dH2O for the gel electrophoresis (0.5 x TAE buffer).
DNA loading buffer
- 50 % (v/v) glycerol
- 1 mM EDTA
- 0.1 % (w/v) bromphenol blue
- Solve in ddH2O
LB medium
For 1 L of LB medium you need:
- 10 g Trypton
- 5 g yeast extract
- 10 g NaCl
- 12 g Agar-Agar (for plates)
- Adjust pH to 7.4
Autoinduction medium for KRX
This medium is based on the LB medium.
After heat sterilization of 900 mL add the following chemicals
- 5 mL of a 200 g L-1 steril L-rhamnose stock solution (final concentration 2 g L-1 L-rhamnose)
- 2.5 mL of a 200 g L-1 steril glucose stock solution (final concentration 0.5 g L-1 glucose)
- if necessary add antibiotics
- Cm: 1 mL of a 20 μg mL-1 Cm stock solution (final concentration 2 μg L-1)
- Amp: 1 mL of a 100 μg mL-1 Amp stock solution (final concentration 100 μg L-1)
- fill-up to 1 L with steril ddH2O
M9 medium
For 250 mL M9 medium you need 175 mL sterile water (for plates add 4 g Agar-Agar as well). Then add (in the following order:
- 250 µL 100 mM CaCl2
- 2.5 mL trace salts
- store this stock solution in the dark
- 250 µL MgSO4
- 250 µL 50 mM FeCl3 / 100 mM citrate (one solution, citrate is iron carrier)
- store this stock solution cold and in the dark
- carbon source stock solution (e.g. glucose)
- 50 mL 5x M9 salts stock solution
- 64 g L-1 Na2HPO4 * 7 H2O
- 15 g L-1 KH2PO4
- 2.5 g L-1 NaCl
- 5 g L-1 NH4Cl
- antibiotic stock solution
- fill up to 250 mL with sterile water
HSG medium
- 14.9 g L-1 glycerine
- 13.5 g L-1 soy peptone
- 7 g L-1 yeast extract
- 2.5 g L-1 NaCl
- 2.3 g L-1 K2HPO4
- 1.5 g L-1 KH2PO4
- 0.249 g L-1 MgSO4 * 7 H2O
5x isothermal reaction buffer for Gibson assembly
storage -20˚C
- 3 mL of 1 M Tris-HCl (pH 7.5)
- 150 µL of 2 M MgCl2,
- 60 µL of 100 mM dGTP
- 60 µL of 100 mM dATP
- 60 µL of 100 mM dTTP,
- 60 µL of 100 mM dCTP
- 300 µL of 1 M DTT
- 1.5 g PEG-8000 and
- 300 µL of 100 mM NAD
Cold osmotic shock buffers for the release of periplasmic protein fraction
Cell fractionating buffer #1 (pH 8)
- 0.2 M Tris
- 200 g L -1 sucrose
- 0.1 M EDTA
Cell fractionating buffer #2 (pH 8)
- 0.01 M Tris
- 0.005 M MgSO4
Denaturation buffer for inclusion bodies
- 6 M urea
- 50 mM Tris-HCl
- 10 mM MgCl2
Buffers for S-layer IEX
Binding Buffer:
- 25 mM Sodiumacetate, pH 6
- 25 mM NaCl
Elution Buffer:
- 25 mM Sodiumacetate, pH 6
- 1 M NaCl
Hanks Buffered Saline Solution (HBSS)
- 0.137 M NaCl
- 5.4 mM KCl
- 0.25 mM Na2HPO4
- 0.44 mM KH2PO4
- 1.3 mM CaCl2
- 1.0 mM MgSO4
- 4.2 mM NaHCO3
Recrystallization buffer SbpA
- 0.5 mM Tris-HCl, pH 9.0
- 10 mM CaCl2
SDS-PAGE gel
The following amouts are for one gel. Stacking gel 5 %:
- 775 μL H2O
- 1.25 mL 0,25 M Tris (pH 6,8)
- 425 μL Bis/Acrylamide (0,8 %, 30 %)
- 50 μL 5 % SDS
- 25 μL 10 % Ammonium persulfate
- 3 μL TEMED
Separating gel 12 %:
- 1.5 mL H2O
- 2.8 mL 1 M Tris (pH 8,8)
- 3.0 mL Bis/ Acrylamide (0,8%, 30%)
- 150 μL 5% SDS
- 37.5 μL 10% Ammonium persulfate
- 5 μL TEMED
SDS running buffer
- 25 mM Tris [pH 8,3]
- 192 mM Glycerol
- 0.1 % SDS
4x Laemmli-buffer
- 250 mM Tris-HCl
- 40 % [v/v] Glycerol
- 20 % [v/v] 2-Mercapthoethanol
- 80 g L-1 SDS
- 0.04 g L-1 BPB
Colloidal Coomassie Brilliant Blue G-250 staining solution
for 1 L staining solution
- dissolve 50 g L-1 (NH4)2SO4 in ddH2O
- add 10 % (v/v) ethanol
- dissolve 0.2 g L-1 Coomassie Brilliant Blue G-250
- add 2 % (v/v) phosphoric acid
- fill up to 1 L with ddH2O
Silver staining solutions
Fixation solution:
- 50 % (v/v) ethanol
- 12 % (v/v) acetic acid
- 1 mL L-1 formaldehyde (37 %)
Thiosulfate solution:
- 0.1 g L-1 Na2S2O3
Impregnation solution:
- 2 g L-1 silver nitrate (AgNO3)
- 0.75 mL L-1 formaldehyde (37 %)
Developing solution:
- 120 g L-1 sodium carbonate (Na2CO3)
- 1 mL L-1 formaldehyde (37 %)
Stop solution:
- 18.6 g L-1 EDTA
Enzyme buffer
- 10 mM MgCl2
- 50 mM Tris/HCl
- pH 7.5
NPI-10 (lysis buffer)
- 50 mM NaH2PO4, pH 8.0
- 300 mM NaCl
- 10 mM Imidazole
- 2 mM PMSF
NPI-20 (wash buffer)
- 50 mM NaH2PO4, pH 8.0
- 300 mM NaCl
- 20 mM Imidazole
NPI-500 (elution buffer)
- 50 mM NaH2PO4, pH 8.0
- 300 mM NaCl
- 500 mM Imidazole
Deadenylation buffer
- 20 mM Tris-HCl, pH 7.5
- 50 mM NaCl
- 4 mM MgCl2
- 1 mM EDTA
- 1 mM DTT
- 10 mM NMN
DNA ligase buffer
- 20 mM Tris-HCl, pH 7.5
- 50 mM NaCl
- 20 % [v/v] Glycerol
NAD+ bioassay buffer
- 50 mM Tris-HCl, pH 8.0
- 10 mM MgCl2
- 2.5 mM CaCl2
- 5 mM DTT
- 0.05 % BSA
Buffers for His-Tag affinity chromatography
- For denaturing conditions add 6 M Urea
- Adjust pH to 7.4 - 7.6
Buffer | Sodium phosphate [mM] | NaCl [mM] | Imidazole [mM] |
---|---|---|---|
Binding buffer | 20 | 500 | 5 |
Elution buffer 1 | 20 | 500 | 40 |
Elution buffer 2 | 20 | 500 | 60 |
Elution buffer 3 | 20 | 500 | 100 |
Elution buffer 4 | 20 | 500 | 300 |
Elution buffer 5 | 20 | 500 | 500 |
Used chemicals
Chemical | Producer | Purity |
---|---|---|
Acetonitrile | VWR | 99.9 %, HPLC Grade |
Benzonase | 20 % | |
Bisphenol A | [http://www.sigmaaldrich.com/catalog/Lookup.do?N5=All&N3=mode+matchpartialmax&N4=bisphenol+a&D7=0&D10=bisphenol+a&N1=S_ID&ST=RS&N25=0&F=PR Sigma] | 97 % |
Bisphenol F | [http://www.alfa.com/de/GP100W.pgm?DSSTK=A11417&rnd=097380668 Alfa Aesar] | 98 % |
Ethylacetate | VWR | > 99.5 %, p.a. |
Isopropyl β-D-1-thiogalactopyranoside | [http://www.carlroth.com/catalogue/catalogue.do;jsessionid=D5D58FA281A3E75342D9198F07546935?id=17413&favOid=000000010000911300020023&act=showBookmark&lang=de-de&market=DE Roth] | ≥ 99 % |
L-rhamnose | [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=de&N4=83650|FLUKA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Fluka] | ≥ 99 % |